Apoptosis is an essential cellular death program that controls proper development and maintains homeostasis. Aberrant regulation of apoptosis contributes to many diseases, such as neurodegeneration, autoimmune diseases, and cancer. The mitochondria-dependent pathway is a major apoptotic pathway, and is primarily regulated and executed by the Bcl-2 family proteins. The Bcl-2 family includes five anti-apoptotic members, two effector proteins Bax and Bak, and eight pro-apoptotic BH3-only proteins. They control the mitochondrial pathway at the step of mitochondrial outer membrane permeabilization (MOMP), a central control point leading to apoptosis. Our long term goal is to elucidate the signaling pathways and molecular mechanisms responsible for mitochondria-dependent apoptosis, and provide positive impact on the development of more potent and specific therapies against apoptosis-related diseases. While genetic and biochemical studies have long established the role of Bax and Bak as two essential effectors of MOMP, the mechanism of Bax/Bak activation, commonly considered the life-to-death switch of the cells, has been intensively investigated in the past two decades. The current consensus is that while all pro-apoptotic BH3-only proteins suppress the anti- apoptotic Bcl-2 proteins, a subset of BH3-only proteins directly engage and activate Bax and Bak during apoptosis. However, in our preliminary studies, we provide genetic evidence suggesting that such a BH3-only protein-mediated direct activation is not necessary for Bax/Bak activation and apoptosis. Instead, our results suggest that upon the BH3-only protein-mediated neutralization of the anti-apoptotic Bcl-2 proteins, Bax/Bak undergo a membrane-dependent, spontaneous activation process. Based on our preliminary studies, we propose a new model of Bax/Bak activation, which will be examined primarily by genetics, cell biology, and biochemical approaches. The following three Aims are proposed. In Aim 1, we will examine the role of the BH3-only proteins and other potential direct activators in Bax/Bak activation following the inactivation of anti- apoptotic Bcl-2 proteins. In Aim 2, we will investigate the involvement of mitochondrial outer membrane in the regulation of Bax/Bak activation. In Aim 3, we will investigate the mechanism of anti-apoptotic Bcl-2 protein- mediated suppression of the Bax/Bak activation. Overall, our proposed studies are expected to elucidate the mechanism of Bax/Bak activation during apoptosis. This proposal may not only unravel one of the long standing mysteries in apoptosis research, but also provide novel targets for therapeutic intervention.